The detection of low-levels of biological and inorganic materials in biological samples, in the body or the environment is frequently difficult. Assays for this type of detection involve multiple steps which can include binding of a primary antibody, several wash steps, binding of a second antibody, additional wash steps, and depending on the detection system, additional enzymatic and washing steps. Such assays further suffer from lack of sensitivity and are subject to inaccuracies. For instance, traditional immunoassays miss detecting 30% of infections.
Molecular probe assays, although sensitive, require highly skilled personnel and knowledge of the nucleic acid sequence of the organism. Both the use of nucleic acid probes and assays based on the polymerase chain reaction (PCR) can only detect nucleic acid which require complicated extraction procedures and may or may not be the primary indicator of a disease state or contaminant. Both types of assay formats are limited in their repertoire in cases where little information is available for the entity to be detected.
Current noninvasive means to measure a patients physical parameters such as CAT or MRI, are expensive and are often inaccessible. Thus, the monitoring of many medical problems still requires tests, which can be slow and expensive. The time between the actual test and the confirmation of the condition may be very important. For example, in the case of sepsis, many patients succumb before infection is confirmed and the infecting organism identified, thus treatment tends to be empirical and less effective. Another example is in screening the blood supply for pathogens.
Verification of a pathogen free blood supply requires a number of labor intensive assays. In the case of HIV-1, the virus that causes AIDS, the current assays screen for anti-HIV antibodies and not the virus itself. There is a window lasting up to many weeks after exposure to the virus in which antibodies are not detectable, and yet the blood contains large amounts of infectious virus particles. Clark et al., 1994, J. Infect. Dis. 170:194-197; Piatak et al., 1993, Aids Suppl. 2: S65-71.
For example, in order to verify that a blood supply is free of HIV-1, several labor-intensive, expensive tests must be performed. Moreover, tests currently in use for initial screening do not identify the virus itself, which can be present at relatively low levels, but are directed to HIV antibodies which are not present for weeks after an initial infection. Clark et al., 1994, J. Infect. Dis. 170:194-197; Piatak et al., 1993, Aids Suppl. 2:S65-71. Thus, screening of the blood supply is not only time-consuming and slow, it may also be inaccurate.
Similarly, the ability to detect substances in the environment, such as airborne and waterborne contaminants is of great importance. For example, it would be desirable to monitor groundwater, to control industrial processes, food processing and handling in real-time using an inexpensive versatile assay. However, current methods) are not suited for such “on-line” monitoring.
There are several reasons why current methods are limited. First, access to sufficient amounts of the material to be detected may be difficult. For example, the detection of biological materials can be difficult as the biological materials of interest are often sequestered inside a body, and large quantities can be difficult to obtain for ex vivo monitoring. Therefore, sensitive assays for use on small amounts of material are necessary. This indicates that a method of amplifying the signal is required. Amplification methods have been established for detection of nucleic acid but this is not the case for antigen detection methods.
A second problem is that sensing may be difficult in real-time because the target materials may be present in small quantities that detection of their presence requires time-consuming, expensive and technical by-involved processes. For example, in the case of bacterial infections in the blood, sepsis, there may be only 1-2 bacteria in a 1-10 ml blood sample. Current methods require that the bacteria are grown first in order to be detected. Askin, 1995, J. Obstet. Gynecol. Neonatal. Nurs. 24:635-643. This time-lag may be detrimental as delaying treatment or mistreating diseases may mean the difference between life and death.
Others have attempted to avoid these limitations by using radioactive or fluorescent tags in combination with antibodies (Harlow et al., (1988), Antibodies. A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press). Antibody-based assays typically involve binding of an antibody to the target molecule, followed by a series of washing steps to remove all unbound antibodies. Binding of the antibody to its target molecule is typically detected by an identifier molecule, for example a secondary antibody specifically recognizing the target molecule specific antibody which contains a detectable label. The step is also followed by multiple wash steps. Alternatively, the target-specific antibody may directly be attached to a detectable label. Labels have included radioactive tracers, fluorescent tags, and chemiluminescent detection systems. Harlow and Lane, 1988, Antibodies. A Laboratory Manual (Cold Spring Harbor, N.Y.: Cold Spring Harbor Laboratory Press).
The series of steps required using such antibody-based assays to generate a specific signal are time consuming and labor intensive. Furthermore, these type of assays are limited to the detection of antigens fixed to some type of matrix. Examples of this type of detection system include Western blots, immunohistochemistry, and ELISA. The highest sensitivity is currently being achieved using radioisotopic and chemiluminescent tags. However, sensitivity, i.e., specific signal over background, of these detection systems frequently remains a limiting factor.
Similarly, background radiation places limits on the sensitivity of radioactive immunoassay techniques. In addition, these techniques are time-consuming and expensive. Finally, radioactive approaches are hostile to the environment, as they present significant waste disposal problems.
Another approach to monitoring substances involves the use of light. Light has the advantage that it is easily measurable, noninvasive and quantitative. Von Bally a al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag).
Traditional spectroscopy involves shining light into substances and calculating concentration based upon the absorbance or scattering of light. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Optical techniques detect variations in the concentration of light-absorbing or light scattering materials. Von Bally a al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Near-infrared spectroscopy has proved to be a nonionizing, relatively safe form of radiation that functions well as a medical probe as it can penetrate into tissues. Further, it is well-tolerated in even large dosages. For example, light is now used to calculate the concentration of oxygen in the blood (Nelicor) or body (Benaron image), or even to monitor glucose in the body (Sandia). Benaron and Stevenson, 1993, Science 259:1463-1466; Benaron et al., 1993, in: Medical Optical Tomography: Functional Imaging and Monitoring, G. Muller, B. Chance, R. Alfano and e. al., eds. (Bellingham, Wash. USA: SPIE Press), pp. 3-9; Benaron and Stevenson, 1994, Adv. Exp. Med. Biol. 361:609-617. However, current techniques are limited in that many substances do not have unique spectroscopic signals which can be optically assessed easily and quantitatively. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag). Furthermore, the detection of substances at low concentration is frequently hampered by high background signals, especially in biological media such as tissues. Von Bally et al., (1982), Optics in Biomedical Sciences: Proceedings of the International Conference (Berlin, N.Y.: Springer-Verlag).
Over the past years, assays based on light emission, for example chemiluminescence (Tatsu and Yoshikawa, 1990, Anal. Chem. 62:2103-2106), have attracted increasing attention due to the development of extremely sensitive methods for detecting and quantifying light. Hooper et al., 1994, J. Biolumin. Chemilumin. 9:113-122. One example of a biomedical research product using chemiluminescence is the ECL detection system (Amersham) for immunoassays and nucleic acid detection.
The use of biological sources of light, bioluminescence, for biological assays has paralleled development of chemiluminescent detection, as similar devices for light detection are required. Kricka, 1991, Clin. Chem. 37:1472-1481. One of the most commonly employed biological source of light is luciferase, a light-generating enzyme synthesized by a range of organisms, including Photinus pyralis (American firefly), Renilla reniformis (phosphorescent coral), and Photobacterium (Luminescent bacterial species). Generally, luciferase is a low molecular weight oxidoreductase, which catalyzes the dehydrogenation of luciferin in the presence of oxygen, ATP and magnesium ions. During this process, about 96% of the energy released appears as visible light. For review, see, Jassim et al., 1990, J. Biolumin. Chemilumin. 5:115-122.
The sensitivity of photon detection and the ability to engineer bacteria and other cells to express bioluminescent proteins permit the use of such cells as sensitive biosensors in environmental studies. Guzzo et al., 1992, Toxicol. Lett. 64:687-693; Heitzer et al., 1994, Appl. Environ. Microbiol. 60:1487-1494; Karube and Nakanishi, 1994, Curr. Opin. Biotechnol. 5:54-59; Phadke, 1992, Biosystems 27:203-206; Selifonova et al., 1993, Appl. Environ. Microbiol. 59:3083-3090. For example, Selifonova et al. describe biosensors for the detection of pollutants in the environment. More specifically, using fusions of the Hg(II) inducible Tn21 operon with the promoterless luxCDABE from Vibrio fischeri, highly sensitive biosensors for the detection of Hg (II) have been constructed.
In addition to systems where bioluminescence is used as detection method of a specific condition, e.g., the presence of Hg(II), supra, constitutive expression of luciferase has been employed as marker to track viability of bacterial cells, as the luciferase assay is dependent on cell viability. For example, constitutive expression of luciferase has recently been employed for the development of drugs and vaccines directed against bacterial disease. Specifically, using an enhanced luciferase-expressing Mycobacterium tuberculosis strain has been employed to evaluate antimicrobacterial activity in mice. Hickey et al., 1996, Antibacterial Agents and Chemotherapy 40:400-407.
However, biosensors that rely on a bacterial receptor to film on a luciferase are limited to sensing those molecules that are have a corresponding bacterial receptor, linked to a known promoter region which can be fused to the luciferase gene. Further, the luciferase-expressing bacteria used to test antimicrobial activity in mice are nonspecific.
Thus, while methods have been explored using the bioluminescence in general, and luciferase in particular, as bioluminescent sensors for very specific applications, the present invention is directed to highly sensitive and highly selective ligand-specific biodetectors for a very broad range of applications. More specifically, the present invention combines the selectivity of ligand-specific binding and the versatility of the antibody repertoire with the sensitivity of bioluminescent detection, employing entities that specifically respond with photon emission to predetermined ligands. The approach of the present invention thus permits the generation of extremely sensitive biodetectors for the development of a wide variety of assays detecting any number of commercially important molecules.